Characterization of Active Anthocyanin Degradation in the Petals of Rosa chinensis and Brunfelsia calycina Reveals the Effect of Gallated Catechins on Pigment Maintenance

Abstract

Anthocyanin degradation decreases ornamental or nutritional values of horticultural products. To investigate factors that may influence colour change in flower development, anthocyanin degradation was compared between the flowers of <i>Brunfelsia calycina</i> and <i>Rosa chinensis</i>, which show rapid and slow degradation, respectively. In-gel activity assays, high performance liquid chromatography (HPLC) analysis of tannins, enzyme kinetics measurement and immune-detection of anthocyanin degradation related-perioxidases (PODs) were carried out for the comparison. Rose petals possessed significantly lower anthocyanin degradation-related POD activities than <i>Brunfelsia</i> petals, which may be related to the high tannin contents. Epicatechin gallate (ECG) and gallocatechin gallate (GCG) were detected in rose as 161.3 ± 12.34 and 273.56 ± 41.23 μg/g FW (Fresh Weight) respectively, while not detected in <i>Brunfelsia</i>. ECG and GCG inhibited the activities of the <i>Brunfelsia</i> POD with half maximal inhibitory concentrations (IC50s) as 21.5 and 29.7 μM respectively, and increased the colour intensities of the anthocyanins. Catechin and epicatechin did not inhibit the POD activity, while serving as POD substrates, with <i>K</i>m (the Michaelis constant) as 0.48 and 1.23 mM. Similar protein levels of the anthocyanin degradation-related 40-kDa PODs were detected in <i>Brunfelsia</i> and rose. In summary, high amount of tannins, particularly ECG and GCG, in red rose petals may inhibit the degradation-related enzymes, leading to the maintenance of anthocyanins in vivo.